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Relief from Zmp1-Mediated Arrest of Phagosome Maturation Is Associated with Facilitated Presentation and Enhanced Immunogenicity of Mycobacterial Antigens

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1556-6811/11/$12.00 doi:10.1128/CVI.00015-11

Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Relief from Zmp1-Mediated Arrest of Phagosome Maturation Is

Associated with Facilitated Presentation and Enhanced

Immunogenicity of Mycobacterial Antigens

Pål Johansen,

1

Antonia Fettelschoss,

1

Beat Amstutz,

2

Petra Selchow,

2

Ying Waeckerle-Men,

1

Peter Keller,

2

Vojo Deretic,

3

Leonhard Held,

4

Thomas M. Ku

¨ndig,

1

Erik C. Bo

¨ttger,

2,5

and Peter Sander

2,5

*

Department of Dermatology, University Hospital Zurich, Zurich, Switzerland1; Institute of Medical Microbiology, University of Zurich,

Zurich, Switzerland2; Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center,

Albuquerque, New Mexico3; Division of Biostatistics, Institute for Social and Preventive Medicine, University of Zurich,

Zurich, Switzerland4; and Nationales Zentrum fu¨r Mykobakterien, Gloriastrasse 30/32, 8006 Zurich, Switzerland5

Received 27 December 2010/Returned for modification 3 February 2011/Accepted 28 March 2011

Pathogenic mycobacteria escape host innate immune responses by blocking phagosome-lysosome fusion. Avoiding lysosomal delivery may also be involved in the capacity of mycobacteria to evade major histocom-patibility complex (MHC) class I- or II-dependent T-cell responses. In this study, we used a genetic mutant of

Mycobacterium bovisBCG that is unable to escape lysosomal transfer and show that presentation of mycobac-terial antigens is affected by the site of intracellular residence. Compared to infection with wild-type BCG, infection of murine bone marrow-derived dendritic cells with a mycobacterial mutant deficient in zinc metal-loprotease 1 (Zmp1) resulted in increased presentation of MHC class II-restricted antigens, as assessed by activation of mycobacterial Ag85A-specific T-cell hybridomas. Thezmp1deletion mutant was more immuno-genicin vivo, as measured by delayed-type hypersensitivity (DTH), antigen-specific lymphocyte proliferation, and the frequency of antigen-specific gamma interferon (IFN-)-producing lymphocytes of both CD4 and CD8 subsets. In conclusion, our results suggest that phagosome maturation and lysosomal delivery of BCG facilitate mycobacterial antigen presentation and enhance immunogenicity.

Mycobacterium tuberculosis is a pathogen with one of the highest death tolls, and its morbific success is in part ascribed to the bacterium’s ability to manipulate host defense mechanisms by evading or controlling host immune responses. One hallmark ofM. tuberculosisinfection is that the bacterium resists lyso-somal delivery following phagocytosis (30). Phagolysosomes are equipped with the machinery to generate peptide-MHC class II complexes (39), and inhibition of phagosome-lyso-some fusion is one proposed mechanism by whichM. tuber-culosismay escape efficient MHC class II antigen presentation (25). Arrest of phagosome maturation may also affect cross-presentation of mycobacterial peptides via the putative phago-some-to-cytosol MHC class I antigen presentation pathway (21) or via MHC class I presentation that occurs by fusion and fission of phagosomes with endoplasmic reticulum-derived ves-icles containing newly synthesized MHC I molecules (27).

BCG is a live attenuated vaccine derived from Mycobacte-rium bovis. More than four billion doses of the vaccine have been administered worldwide since its introduction in 1921. Although relatively safe and inexpensive, the efficacy of BCG to protect against adult lung tuberculosis is highly variable (7). The reasons for the poor efficacy of BCG in protection against tuberculosis are poorly understood. One explanation builds on the observation that BCG has lost important genes during the laboratory attenuation process (2). As a consequence, much

work has been done to improve, with varying success, the efficacy of BCG by introducing additional copies of existing genes (14) or by reintroducing some of the genes that were lost during thein vitroattenuation process (26).

It is widely assumed that tuberculosis disease will protect, at least partially, against subsequent reinfection (1). However, the failure of natural disease to protect against reinfection (36) indicates that immunity evoked by natural infection is limited, partially explaining the relative ineffectiveness of vaccination with BCG (24). The limited postinfection immunity may also indicate thatM. tuberculosisactively escapes immune surveil-lance (12, 25, 28). We have previously reported that a putative mycobacterial zinc metalloprotease, Zmp1, plays an important role in disease pathogenesis by interfering with inflammasome activation and phagosome maturation (23), two central pathways of pathogen defense. In the present study, we hypothesized that the limited efficacy of BCG reflects the natural course of tuberculosis infection and the organism’s ability to subvert the immune system. Consequently, rather than attempting to im-prove BCG efficacy by introducing additional genes, we fol-lowed the idea that further genetic deletions might increase BCG immunogenicity.

Processing and presentation of mycobacterial antigens are impaired in mycobacterial infections (30), and it is important to determine whether this is connected with phagosome mat-uration. Pursuing the hypothesis that the mechanism through which a vaccine is processed in antigen-presenting cells affects its efficacy, we speculated thatzmp1deletion might alter BCG immunogenicity. Using anM. bovis BCG mutant lacking the zmp1 gene, we assessed the possible influence of Zmp1 on * Corresponding author. Mailing address: Institute of Medical

Mi-crobiology, Gloriastrasse 30/32, 8006 Zurich, Switzerland. Phone: 41 44 634 2684. Fax: 41 44 634 4906. E-mail: psander@imm.uzh.ch.

Published ahead of print on 6 April 2011.

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presentation of mycobacterial antigens by professional anti-gen-presenting cells and the induction of CD4- and CD8-pos-itive T-cell responses to mycobacterial antigens in vivo. By various measures of immune reactivity, we show that Zmp1 deletion increased the immunogenicity of BCG both in vitro andin vivo.

MATERIALS AND METHODS

Mice.Female BALB/c (H-2d

) or C57BL/6 (H-2b

) mice were obtained from Harlan (Horst, Netherlands). Female SCID (CB-17/lcr-PrkdcSCID) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). All mice were ob-tained and kept under specific-pathogen-free (SPF) conditions in facilities at the University Hospital Zurich and were used at 6 to 10 weeks of age. The experi-ments were reviewed by the local ethical committee and were performed accord-ing to Swiss experimental and ethical guidelines.

Preparation and cultivation of bacteria.Mycobacterial strains were grown and propagated according to standard microbiological techniques. The con-struction of the BCGzmp1knockout mutant has been described previously (23). BCGzmp1was complemented by transformation with thezmp1 -contain-ing plasmid, pMV361-hyg-zmp1, and the complemented BCG strain is hence-forth referred to as BCGcompl. Zmp1 expression in the complemented mutant is under the control of thezmp1promoter.

Western blot analyses using rabbit anti-Ag85 antiserum (gift from Colorado State University) showed comparable expression levels for Ag85 in all three strains: BCG, BCGzmp1, BCGcompl(data not shown).

Confocal imaging.Bone marrow stem cells were isolated from mice femurs and differentiated for 7 days in Dulbecco’s modified Eagle’s medium supple-mented with 10% fetal calf serum (FCS), 10% L cell conditioned medium, and penicillin/streptomycin on petri dishes. The cells were then mounted on 0.7-mm glass coverslips in 24-well plates at 1.5⫻105to 2105cells per well and infected with BCG as described previously (29). Colocalization studies were done as described previously (29), and the coverslip contents were left unidentified be-fore analysis.

Antigen presentation studies in dendritic cells.Dendritic cells (DCs) were prepared from C57BL/6 and BALB/c mice as described previously (17). Briefly, femurs were aseptically harvested and bone marrow cells were cultured in RPMI 1640 medium supplemented with 10% FCS, glutamine, sodium pyruvate, peni-cillin, and streptomycin in the presence of 10% supernatant from granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting X-63 cells. The X-63 cell line was transfected and kindly provided by A. Rolink (University of Basel). After 6 to 7 days, 1⫻105

DCs per well were plated in 96-well flat-bottom culture plates and infected with BCGwt, BCGzmp1, or BCGcomplat different multiplic-ities of infection (MOIs) (10, 1, 0.1, 0.01, or 0). Fluorescence-activated cell sorter (FACS) analyses of DCs infected with Alexa Fluor 488-labeled mycobacteria demonstrated comparable mean fluorescence levels for BCGwt, BCGzmp1, and BCGcompl, and fluorescence microscopy of macrophages showed similar infec-tion rates for all three strains (data not shown). Within 1 h, 1⫻105

DE10 (H-2b

) or 2E5 (H-2d) T-cell hybridoma cells specific for theM. tuberculosisAg85A

peptides comprising amino acids 241 to 260 [Ag85A(241–260)] and amino acids 101 to 120 [Ag85A(101–120)], respectively, were added to each well. The anti-gens were purchased from EMC (Tu¨bingen, Germany), and the hybridomas were kindly provided by C. Leclerc (Institut Pasteur, Paris). After incubation at 37°C for 20 h, supernatants were collected and frozen for later analysis of interleukin-2 (IL-2) by standard enzyme-linked immunosorbent assay (ELISA) (R&D Sys-tems, Abingdon, United Kingdom).

Analysis of delayed-type hypersensitivity.C57BL/6 mice were immunized with different doses of BCGwtor BCGzmp1 in 100␮l phosphate-buffered saline (PBS) by subcutaneous injections in the neck region. After 3 weeks, the mice were challenged by injection of 50␮l of a 5-␮g/ml solution of tuberculin purified protein derivative (PPD) (SSI, Copenhagen, Denmark) in saline, into the plantar side of the hind right footpad. Injection of saline alone was used as a negative control. One to 4 days later, a delayed-type hypersensitivity (DTH) reaction was analyzed by measuring the swelling of the footpad in comparison to the prechal-lenge thickness of the footpad. The measurements were done using a spring-loaded digital micrometer from Mitutoyo (Kawasaki, Japan).

Immunogenicity testing. C57BL/6 mice were immunized by subcutaneous (s.c.) injection of 106

CFU BCG on days 0 and 21. On day 28, the mice were euthanized and spleens harvested. The spleens were homogenized, the erythro-cytes were lysed in a hypotonic buffer, and triplicates of 2⫻105

splenocytes were restimulatedin vitrowith 5 or 1␮g/ml PPD for determination of proliferation and cytokine secretion. After 3 days of cultivation, supernatants were collected

and frozen for later analysis of IFN-␥secretion by ELISA (R&D Systems). Parallel cultures were pulsed with3

H-labeled thymidine at 1␮Ci per well for another 16 h for analysis of proliferation by␤-scintillation.

IFN-␥-producing cells were analyzed by enzyme-linked immunospot (ELISPOT) assay (Diaclone, Besanc¸on, France) by restimulating 2⫻105

splenocytes from immunized mice with or without antigen for 16 h in multiscreen 96-well Millipore polyvinylidene difluoride (PVDF) plates (Fisher Scientific AG, Wohlen, Swit-zerland) precoated with 1␮g/ml anti-IFN-␥. The ELISPOT plates were then washed and developed according to the manufacturer’s protocol. The spots were analyzed on an AID EliSpot reader system from Autoimmun Diagnos-tika (Strassberg, Germany), and the results were expressed as spots per 2⫻105 splenocytes.

IFN-␥-producing splenocytes were analyzed by intracellular staining and flow cytometry. Triplicates of 2⫻106splenocytes were restimulated in 24-well plates at 37°C for 16 h with 5␮g/ml PPD, the last 4 h also with 2.5␮g/ml brefeldin A (Sigma-Aldrich, Buchs, Switzerland). The cells were washed, fixed in protein-free PBS/paraformaldehyde (1%) on ice for 10 min, washed, and then permeabilized in PBS/NP-40 (0.1%) on ice for 3 min. After washing, the cells were resuspended in PBS/FCS (2%), incubated on ice for 5 min with anti-CD16/CD32 for Fc-receptor blocking, and then stained with anti-CD4 (fluorescein isothiocyanate [FITC]), CD8 (PerCP-Cy5), CD44 (phycoerythrin [PE]), and anti-IFN-␥(allophycocyanin [APC]) antibodies for 40 min. After washing, the cells were acquired using a FACSCanto (BD Biosciences, San Jose, CA). All anti-bodies were purchased from BD Pharmingen (Basel, Switzerland) or from eBio-science (Bender MedSystems, Vienna, Austria). The analysis was done using the FlowJo 8.5.2 software from Tree Star, Inc. (Ashland, OR), and the frequency of IFN-␥-producing cells was defined by gating on CD44-positive CD4 or CD8 lymphocytes.

Persistence of BCG in immunocompetent mice.The effect of Zmp1 on the growth of BCG in immunocompetent mice was tested after tail vein injection of 106

CFU of BCGwtor BCGzmp1in C57BL/6 mice (n⫽40). At different time points thereafter, 5 to 7 animals from each group were euthanized, and the lungs, spleen, and frontal liver lobe of each animal were harvested aseptically in 5 ml PBS. The tissues were homogenized using a Polytron PT 3000 from Kinematica (Littau, Switzerland), incubated for 1 h in 0.5% quillaja bark saponin (Sigma-Aldrich), serially diluted, and plated on Middlebrook 7H10 plates for the deter-mination of the numbers of BCG bacteria. The colonies were enumerated after 3 weeks of incubation at 37°C.

Pathogenicity of BCG in SCID mice.To test whether the deletion of Zmp1 in BCG affects its pathogenicity in immunodeficient mice, the survival of SCID mice upon inoculation with BCGwtand BCGzmp1was assessed. The two BCG strains were administered by tail vein injections at 106

, 107 , and 108

CFU in groups of six SCID mice. Body weight and survival were assessed over a period of 6 months. The animals were euthanized when they met the preestablished endpoint of 20% weight loss compared to the weight at the day of inoculation.

Statistical analysis.For the analysis of dependent observations, a random-effects analysis of variance (ANOVA) was performed, with the BCG strain, immunization dose, and type of antigen used for restimulation as fixed factors and the individual mouse as a random effect. Prior to ANOVA, the data were square root transformed to meet the equal variance assumption. Nonparametric and independent data were analyzed by the two-sided Mann-Whitney U test for two groups or by the Kruskal-Wallis test for three groups or more. For analysis of the survival data, mean survival time and hazard ratios (with 95% confidence intervals) were computed. Kaplan-Meier survival curves were compared with the use of the Cox proportional-hazards models, and statistical significance was tested by log rank test. Analyses were done using the statistical software R (30) or GraphPad Prism. The significance level was set at 5%.

RESULTS

Zmp1-dependent phagosome maturation arrest. At early stages, the course ofM. tuberculosisinfection in macrophages is characterized by the bacterially induced arrest of phago-somal maturation. This allows the bacterium to replicate in vacuoles that retain a high pH, show limited hydrolytic activity, and intersect poorly with antigen presentation pathways (30). BCGwtarrests phagosome maturation at the stage of an early endosome. However, deletion ofzmp1suspends the ability of the bacteria to arrest phagosome maturation, and BCGzmp1

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izes with lysosome-associated membrane protein (LAMP-1), a marker for late endosomes (Fig. 1).

In vitroantigen presentation of BCG.Zmp1 interferes with innate immune responses (23), which prompted us to investi-gate whether the deletion of Zmp1 affects mycobacterial anti-gen presentation in professional antianti-gen-presenting cells. Bone marrow DCs were prepared and infected at different MOIs with BCGwt, BCGzmp1, or BCGcompl. The infected DCs were incubated with MHC class II-restricted T-cell hybridomas DE10 and 2E5 specific for the mycobacterial antigen epitopes Ag85A(101–120) (I-Ed) and Ag85A(241–260) (I-Ab),

respec-tively, and the secretion of IL-2 from the hybridoma cells was measured as a marker of antigen-stimulated T-cell activation. Presentation of Ag85A antigens was found to be more efficient in cells infected with the Zmp1-deficient BCG strain than in cells infected with the wild type or the complemented strains (Fig. 2). Increased antigen presentation in BCGzmp1-infected cells was independent of MHC restriction, as it was observed in

DCs derived from bothH-2bC57BL/6 (Fig. 2A) (P0.04) and H-2dBALB/c (Fig. 2B) (P0.0001) mice.

Immunogenicity of the BCG wild type and Zmp1 mutants.

To analyze the role of Zmp1 in the immunogenicity of BCG, mice were immunized with titrated doses of wild-type and Zmp1-deficient strains. Subsequently, we measured T-cell re-sponses by assessing DTH to PPD injections into footpads. Different thresholds for the two strains were observed. While inoculation of 104CFU BCGwtwas required to produce

no-table and specific DTH to PPD, 103 CFU was sufficient to

induce an equivalent response with BCGzmp1 (Fig. 3A). BCGzmp1induced a significantly stronger DTH response than did BCGwtat 103CFU (P0.0004), while the difference at

104CFU (P0.08) was of borderline significance (Fig. 3A).

The peak of footpad swelling was observed with inoculations of between 104 and 105 CFU (data not shown). Data from a

second independent experiment were available, and the anal-ysis of the combined data (n ⫽ 78) verified a significantly stronger DTH response at both 103(P0.0001) and 104(P

0.04) CFU. When the kinetics of the DTH response were analyzed for inocula of 104CFU BCG, the results revealed that

BCGzmp1produced stronger and faster DTH responses than BCGwt, the maximum swelling being reached 48 h earlier for BCGzmp1than for BCGwt(Fig. 3B).

The increased immunogenicity of the Zmp1-deficient and lysosome-targeting BCG strain was associated with increased antigen-specific T-cell proliferation and cytokine secretion of splenocytes from immunized mice. Figure 4A illustrates anti-gen-specific T-cell proliferation after inoculation of 106CFU

BCG and for splenocytes restimulated with PPD. First, while 1

␮g/ml was sufficient for restimulation and proliferation of splenocytes from mice immunized with BCGzmp1, a 5-fold-higher concentration was required for splenocytes harvested from mice immunized with BCGwt or the rescued, comple-mented mutant BCG. In addition, the degree of proliferation was significantly increased following BCGzmp1immunization (P⫽0.02 by ANOVA). The assessment of secreted IFN-␥by ELISA revealed increased cytokine amounts following immu-nization with BCGzmp1 compared to BCGwt (P ⬍ 0.0001) FIG. 1. Intracellular localization of BCG. BCGzmp1 but not

BCGwtcolocalizes with LAMP-1. Representative confocal immuno-fluorescence images of bone marrow-derived macrophages infected with FITC-labeled BCGwt(upper panel) and BCGzmp1(lower panel). FITC-labeled bacteria are green (left). LAMP-1-labeled late endo-somes stain red (middle). Yellow spots or patches in overlaid images (right) indicate BCG located inside phagolysosomes. The cells were stained with LAMP-1 60 min after infection.

FIG. 2.In vitropresentation of mycobacterial Ag85A. Bone mar-row dendritic cells (DCs) were prepared fromH-2bC57BL/6 (A) or H-2bBALB/c (B) mice and infected at various MOIs with BCGwt

(open bars), BCGzmp1 mutant (filled bars), or the zmp1 -comple-mented strain BCGcompl (hatched bars). The infected DCs were cocultured for 20 h with the I-Ab-restricted Ag85A(241–260)-specific T-cell hybridoma DE10 (A) or with the I-Ed-restricted Ag85A(101– 120)-specific T-cell hybridoma 2E5 (B). The IL-2 secretion by the T-cell hybridomas was quantified by ELISA. The data are means plus standard deviations of results for 3 (DE10) and 6 (2E5) replicates. The experiments were performed three times with comparable results. Sta-tistical differences between BCGwtand BCGzmp1were analyzed by a two-way ANOVA, the variables being MOI and BCG genotype.

FIG. 3. DTH reactions in BCG-immunized mice. C57BL/6 mice were immunized with 102, 103, or 104CFU BCGwtor BCGzmp1and challenged intradermally with PPD in the footpad on day 21. (A) Foot-pad DTH was measured 48 h postchallenge (n⫽66). The box plots and whiskers show the minimum, maximum, 25th and 75th percentiles, and median. (B) In a different experiment, footpad DTH was mea-sured 24, 48, and 96 h postchallenge of mice immunized with 104CFU BCGwt(open symbols) or BCGzmp1(closed symbols) (n⫽12). The data are means⫾standard errors of the means.Pvalues were calcu-lated by the Kruskal-Wallis test (A) and ANOVA (B).

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(Fig. 4B). In addition, the number of IFN-␥secreting cells, as determined by ELISPOT, was significantly increased in BCGzmp1-immunized mice compared to that in mice immunized with BCGwt(P⫽0.05, more precisely 0.0476) (Fig. 4C). To analyze whether IFN-␥secretion derived from CD8- or CD4-positive T cells, splenocytes from mice were restimulated with PPD and analyzed by flow cytometry (Fig. 4D). Compared to the results with untreated controls, BCG primed T cells for IFN-␥production (CD4,P⫽0.0005; CD8,P⫽0.0006; Mann-Whitney test). Splenocytes from mice immunized with BCGzmp1contained significantly higher frequencies of IFN-␥ -producing CD4 (P⫽0.004) and CD8 (P⫽0.008) T cells than did splenocytes from mice immunized with BCGwt or BCGcompl, while the two latter strains induced comparable numbers of IFN-␥positive cells.

In additional experiments, mice were immunized as de-scribed above, but the splenocytes were stimulated with myco-bacterial Ag85A or TB10.3 antigens (not shown). A two-way random-effects ANOVA revealed that splenocytes from BCGzmp1-immunized mice had a stronger proliferation ca-pacity (P⫽0.007) and produced more IFN-␥(P⫽0.02) than splenocytes from BCGwt-immunized mice. Here, a three-way random-effects ANOVA applied to all data available (n⫽360 observations from 30 mice) revealed that stronger immune reactions were observed in mice immunized with thezmp1 -deleted mutant than in mice immunized with BCGwt (P

0.0001), independent of thein vivoBCG dose or the type of antigen used for restimulationin vitro.

BCG persistence and growth in wild-type and SCID mice.

To test if deletion ofzmp1and increased phagosome matura-tion is associated with differences in disseminamatura-tion and persis-tence of BCG, wild-type mice were infected with 106 CFU

BCGwtor BCGzmp1intravenously. General signs of disease and the bacterial load of various organs were monitored over a period of 85 days. No overt signs of clinical disease were observed during the experiment. Systemic infection caused a high mycobacterial load in the spleen and liver (Fig. 5). The titers slowly declined, but mycobacteria were still detectable in livers and in spleens nearly 3 months postinfection. The bac-terial load in the lungs was approximately 1% of the load in the liver 1 day postinfection and declined to undetectable levels at the end of the study (not shown). No difference in dissem-ination or persistence was observed between BCGwt and BCGzmp1.

We also addressed the possible unexpected adverse effects ofzmp1deletion by infecting SCID mice and monitoring their survival. The animals received 106, 107, or 108CFU of either

BCGzmp1or the parental BCGwtstrain by tail vein injection, and their survival was monitored. The criterion for termination was a weight loss of 20%, as illustrated in Fig. 6A for an inoculum of 106CFU BCG. The mice started to lose weight

after 5 to 6 weeks following infection and had to be euthanized at approximately 9 to 12 weeks postinfection, independent of Zmp1 expression (Fig. 6A). The Kaplan-Meier survival curves for all inoculum doses revealed dose dependency but strain independency with mean survival times of 9 and 16.5 days (108

CFU), 42.5 and 44 days (107CFU), and 78.5 and 80.5 days (106

CFU) for BCGwtand BCGzmp1, respectively (Fig. 6B). Sta-tistical analysis comprising dose pairwise comparisons of mice infected with BCGzmp1or BCGwtusing log rank and hazard FIG. 4. Analysis of antigen-specific splenocytes. Groups of six

C57BL/6 mice were immunized with 106CFU BCGwt, BCGzmp1, or BCGcomplon days 0 and 21; littermate controls were left untreated. On day 28, splenocytes were harvested and restimulatedin vitrowith 5 or 1␮g/ml PPD or medium. (A) Lymphocyte proliferation ([3 H]thy-midine incorporation) was measured after 4 days, and the stimulation index (SI) was determined against nonstimulated cultures. (B) IFN-␥ secretion was measured in supernatants in 3-day cultures by ELISA. (C) The ELISPOT assay shows the number of IFN-␥-secreting cells per 200,000 splenocytes. (D) The flow cytometry data show the per-centage of IFN-␥-producing cells after gating on CD4- or CD8-positive and CD44-positive lymphocytes. The histograms show means and stan-dard errors of the means, while box plots show the minimum, maxi-mum, 25th and 75th percentiles, and median.Pvalues were obtained from two-way random-effects ANOVA comparing BCGzmp1 with BCGwt(A to C) or by the Mann-Whitney test (D). One out of 2 or 3 representative experiments is shown.

FIG. 5. BCG persistence in wild-type mice. C57BL/6 mice received 106CFU BCGwtor BCGzmp1by intravenous injections (n40). At different time periods postinjection, mice were euthanized and the spleens and livers were harvested and homogenized. Titrated amounts of the tissue homogenates were plated on 7H10 agar plates for deter-mination of the bacterial load. The data are means⫾standard errors of the means. No statistical difference was observed between BCGwt

and BCGzmp1.

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ratio testing revealed no evidence for a difference between the mutant and the parental strain (Fig. 6C).

DISCUSSION

Applied for almost a century, vaccination againstM. tuber-culosiswithM. bovis BCG protects poorly against pulmonary infections in adults. This inefficiency is illustrated by the ob-servation that almost all of the nearly 10 million new patients contracting tuberculosis disease annually had been vaccinated with BCG (41). Consequently, diverse strategies have been explored to improve vaccination (20) but also to understand how M. tuberculosis subverts immune responses. Most ap-proaches to improve BCG vaccination have involved overex-pression of mycobacterial antigens (14, 26). One of the current and perhaps most promising vaccine candidates is a recombi-nant BCG strain that expresses listeriolysin, which facilitates endosomal escape and cytosolic delivery, thereby inducing a shift toward MHC class I-restricted antigen presentation (10, 11).

The present study shows that a genetic deletion of BCG may evoke improved antigen presentation and immunogenicity. Our approach is based on two assumptions. First,M. tubercu-losis has evolved to subvert antimycobacterial immune re-sponses by arresting the maturation of phagosomes (30). As a result, phagosome-lysosome fusion is blocked and efficient MHC class II-mediated antigen presentation is impaired. Sec-ond, BCG has, at least partially, retained these properties. We characterized a BCG mutant that is deficient in the gene encoding zinc metalloprotease 1 (Zmp1), a protein which previously has been shown to inhibit phagosome maturation by preventing inflammasome activation and caspase-depen-dent IL-1␤production (23). Using mycobacterial Ag85A-spe-cific T-cell hybridomas, we found that infection of murine DCs with Zmp1-deficient M. bovis BCG significantly enhanced antigen presentation compared to that of wild-type BCG. This was observed for both I-Ab- and I-Ed-restricted MHC

class II pathways of presentation, as similar data were ob-tained with C57BL/6 (H-2b) and BALB/c (H-2d) mice, respec-tively. Increased antigen presentation was accompanied by en-hanced BCG immunogenicity: (i) the DTH reaction upon immunization with BCGzmp1was obtained with 1/10 of the dose required for BCGwt, (ii) the induction of antigen-specific splenocyte proliferation was increased, (iii) the fre-quency of antigen-specific IFN-␥-producing CD4 and CD8 T cells was higher, and (iv) the amount of IFN-␥secreted was heightened following immunization with BCGzmp1. To di-rectly demonstrate that the increased immunogenicity of the zmp1deletion mutant is due to inactivation of the structural zmp1gene, we complemented thezmp-deficient mutant with wild-typezmp1. Complementation withzmp1abrogated phago-some maturation (23), and the mutants increased immunoge-nicity.

Despite extensive use of BCG, the correlates of protection are still ill defined (19). While activated macrophages are crit-ical for disease containment (6), both hypo- and hyperactiva-tion have been suggested to contribute to disease progression (42). The finding thatzmp1deletion did not affect survival of BCG-infected immunodeficient SCID mice nor BCG growth and dissemination in immunocompetent mice indicates that the enhanced immunogenicity of BCGzmp1does not come at the cost of decreased persistency or heightened pathology. The exact mechanism of mycobacterium-induced phagosome matu-ration arrest is still unclear. The bacteria are taken up by macrophages or DCs and are internalized into phagosomes that fuse with early and late endosomes (4, 31, 33). Phagosome maturation from early phagosomes to phagolysosomes com-prises a series of fusion, fission, and trafficking events, which depend on a complex network of Rab GTPases and phospho-lipids that control endocytic processes (3, 32, 34, 35). Besides its direct antibacterial effects, the lysosome and its acidic en-vironment are vital for antigen presentation through degrada-tion and unfolding of bacterial proteins and the substitudegrada-tion of the invariant chain that allows binding to MHC class II mole-cules (30). In addition to Zmp1, several other proteins and lipids reportedly inhibit phagosome maturation and lysosomal delivery of pathogenic mycobacteria. Cell wall lipids are asso-ciated with the blocking of phagosome-lysosome fusion, e.g., lipoarabinomannan (8, 13, 37), sulfolipids (9), and trehalose dimycolate (15), and the bacterial phosphatase SapM (38), the FIG. 6. BCG testing in SCID mice. SCID mice received BCGwtor

BCGzmp1by intravenous injections (n⫽36). The body weights were measured before injection and individually monitored thereafter. Upon a weight loss of 20%, the animals were euthanized. (A) Weight monitoring of groups of six SCID mice that received 106CFU of either BCG strain. (B) Survival of SCID mice in groups of six receiving 106, 107, or 108 CFU of either BCG strain illustrated by Kaplan-Meier curves. (C) The hazard ratio (HR) and log rank analysis of relative risks demonstrated no statistical difference between BCGwtand BCGzmp1.

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nucleoside diphosphate kinase Ndk (34), and the serine/thre-onine protein kinase G (PknG) (5, 40) have all been suggested to affect the maturation of phagosomes.

Our results are at variance with recent observations that the arrest of phagosome maturation, as mediated by deletion of the serine/threonine protein kinase G (PknG) (5, 40), does not affect the presentation of BCG antigens (22). While we cannot directly explain this discrepancy, subtle, yet unknown differ-ences in phagosome maturation may be responsible for func-tional differences between the Zmp1- and the PknG-deficient mutants. Support for our conclusion that phagosome matura-tion does play a role in presentamatura-tion and immunogenicity of mycobacterial antigens is provided by recent data from studies in which rapamycin was used to pharmacologically enhance trafficking of BCG to lysosomes by means of autophagy. Upon treatment with rapamycin, mycobacteria colocalized with the late endosomal markers Rab7 and CD63, and this result correlated with enhanced antigen presentation (16). In ad-dition, Ag85A-deficient virulentM. tuberculosisbacteria re-side in a phagosomal compartment enriched for the acido-tropic dye LysoTracker and LAMP-1, suggesting enhanced fusion with late endosomes, and this is correlated with im-proved T-cell primingin vitro(18). Together with our results, these studies indicate that phagosome maturation and antigen presentation are interconnected and that the immunogenicity of BCG is affected by triggering phagosome maturation and lysosomal delivery of mycobacterial antigens.

In summary, this study demonstrates that deletion mu-tagenesis of BCG may not only facilitate phagosome matura-tion but also increase antigen presentamatura-tion and immunogenic-ity of BCG. These findings may lead to a better understanding of the molecular mechanisms involved in tuberculosis-associ-ated subversion of the host’s immune system. The results also suggest that deletion of genes that suppress antigen presenta-tion and immunogenicity is a promising approach in the ratio-nal design of new vaccines againstM. tuberculosis.

ACKNOWLEDGMENTS

This project was supported in part by the University of Zurich, the European Union (EU-FP7 New TBVac, project no. 241745), the Swiss National Science Foundation (3100A0-120326), and the Niedersa ¨chsis-cher Verein zur Beka¨mpfung der Tuberkulose, Lungen- und Bronchialer-krankungen e.V. P.J., T.M.K. (3100AO-122221), and Y.W.-M. (3200BO-118202) were supported by the Swiss National Science Foundation.

The authors disclose no conflict of interest.

We thank Nicole Graf for assistance with statistical analysis, Deepa Mohanan for FACS sorting of infected dendritic cells, and Claude Leclerc for the T-cell hybridomas. Anti-Ag85 serum was obtained under NIH contract HHSN266200400091C/ADB, contract NO1-AI-40091, Tuberculosis Vaccine Testing and Research Materials Contract via Colorado State University.

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Figure

FIG. 2. presentation of mycobacterial Ag85A. Bone mar-120)-specific T-cell hybridoma 2E5 (B)
FIG. 6. BCG testing in SCID mice. SCID mice received BCGwtBCGcurves. (C) The hazard ratio (HR) and log rank analysis of relativerisks demonstrated no statistical difference between BCGBCG strain

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